Method for determination of a biologically active substance in an analyzed liquid and device for its realization

ABSTRACT

The present invention allows quickly, simply, precisely and with high sensitivity to determine the presence and concentration of any biologically active substances capable to interact with linear double-stranded DNA molecules in any liquids including biological liquids. The essence of the offered method consists that the lyotropic liquid-crystalline cholesteric DNA dispersion is formed in an aqueous-salted solution of a polymer, neutral in respect to DNA, of the linear double-stranded DNA molecules of a low molecular mass immediately before mixing with an analyzed liquid containing the determined substance, thereat the liquid for the analysis is prepared by mixing with the specified polymer under conditions, when the optical properties of the lyotropic liquid-crystalline DNA dispersion are not broken, then through the sample obtained as a result of mixing of the prepared analyzed liquid with the liquid-crystalline DNA dispersion the circular-polarized light is passed, and the optical signal is registered at two wavelengths, one of them is in the region of the DNA absorption, and another one is in the region of absorption of the biologically active substance, then the ratio between these two signals is calculated, and concentration of the biologically active substance is determined on this ratio using the calibration curve. The offered device contains a wavelength selector ( 2 ) having an electrodynamic driver ( 22 ) of a positional type and at least one optical element ( 26, 27, 28 ) fixed on a motor ( 33 ) shaft ( 35 ) of the specified driver ( 22 ).

FIELD OF THE INVENTION

The present invention concerns to the medical engineering and thepharmaceutical industry, and more particularly—to the way ofdetermination in an analyzed liquid of a biologically active substanceand the device for its realization.

The offered invention can be used in medical and clinical biochemistryand also in the molecular pharmacology at research of pharmaco-kineticsof biologically active substances, in pharmaceutical industry and inecology. The most effective use of the present invention is in clinicalbiochemistry.

DESCRIPTION OF THE RELATED ART

The basic problem at rational pharmaco-kinetics of biologically activesubstances (BAS), in particular, synthetic and half-synthetic antitumorsubstances, influencing a possibilities of effective therapy, is reducedto speed and accuracy of determination of concentration of thesesubstances in biological liquids (blood, plasma of blood, urea, etc.)after treatment of patients with certain amount of an antitumorsubstance.

Two basic groups of methods are known for determination of the presenceand the efficiency of “action” of BAS, basic “target” of which aremolecules of double-stranded nucleic acids.

The first group is formed by the biological methods [Modern methods inbiochemistry. Ed. by V. N. Orekhovich. M.: Medicine. 1968, p.372;Methods of practical biochemistry. Ed. by B. Williams, K. Wilson. M.:World, 1978, p. 256]. They are based on the investigation ofwell-registered changes in well-described genetic systems (bacteria,bacteriophages, cultures of cells) after their treatment with BAS. Thesemethods are realized by means of the standard microscopes. However, on away of BAS penetration, the modification of their structures canHowever, on a way of BAS penetration, the modification of theirstructures can take place influencing the accuracy of the establishmentof correlation between the structure of BAS, its concentration and thebiological activity. In addition, the specified methods differ byduration of realization of the experiment (from days till weeks).

The second group includes the physico-chemicalical methods or theirdifferent combinations. Since 1980, a few methods of determination ofBAS, in particular, derivatives of the anthraquinone group have beenoffered. Among them there are various versions of the radioimmuneanalysis [Nicolau G., Szucs-Myers V., McWilliams W., Morrison J.,Lanzilotti A. (1985). Investigational New Drugs, 3: pp.51-56 ], thehigh-pressure thin-layer chromatography [Avramis V. (1982), Abstract.Pharmacologist, 24, p.241; Ehninger G., Proksch B., Hartmann F., GartnerH. V., Wilms K. (1984). Cancer Chemotherapy and Pharmacology, 12,pp.50-52 ], the method of replacement of DNA bound ethydiumbromide[Horvath J. J., Gueguetchkeri M., Gupta A., Penumatchu D., Weetall H.H., (1995), Biosensor and Chemical Sensor Technology. Ed. by Rogers K.R. et al., Washington, ASC Symp.Ser. 613, pp. 45-60]. The greatestapplication has been gained by the columnarhigh-pressure-liquid-chromatography (HPLC) [Chiccarelli F. S., MorrisonJ. A., Cosulich D. B., Perkinson N. A., Ridge D. N. (1986), CancerResearch, 46, pp. 4858-4861; Lin K. T., Rivard G. E., Leclerc J. M.(1989). J. Chromatography, 465, pp.75-86], carried out by the standardchromatographic devices.

The application of the HPLC method for determination of one of importantanthraquinone—mithoxantrone (MX) has been described rather recently[Nicoletto M. O., Padrini R., Ferrazi E., Nascimben O., Visona E.,Tumolo S., Palumbo M., Cossta L., Vinante O., Monfardini S., FiorentinoM. V. (1993). Eur. J. Cancer. 29A, pp.1242-1248]. According to thismethod, MX and products of its methabolism are extracted from blood ofpatients, then they are concentrated, and the chromatographic allocationof MX is carried out with a subsequent spectrophotometric determinationof its concentration. Despite rather high accuracy of MX determination(a few tens of ng/ml), the specified method is characterized by:

duration of the whole determination process reaching two days, that iscaused by the necessity of a special pretreatment of samples (processingof patients blood, extraction and concentration of MX, etc.),

application of rather expensive equipment, namely, the high-pressurechromatographs or similar devices,

necessity to use the high skilled personnel for realization of the wholecycle of the analysis.

A laboratory method for determination of colored BAS, those “targets”are the double-stranded DNA molecules, that takes into account theinteraction of BAS with DNA molecules forming liquid crystalsimmobilyzed in films (gels) biosensor is known as well [Skuridin S. G.,Pozdnyakov V. N., Tokareva L. G., Yevdokimov Yu. M. (1991), Patent ofthe Russian Federation N 2016888].

This method includes:

formation of the lyotropic liquid-crystalline dispersion of DNA in anaqueous-salt solution containing a neutral polymer,

addition the special monomers capable to be polymerized, andpolymerization of the obtained mixture,

reception of a film (gel) with the form and the size that are convenientfor the experimentator,

immersing of the film (gel) into an analyzed laboratory solutioncontaining BAS, and exposure of the film in this solution during timethat is sufficient for diffusion of BAS into the film and interactionwith DNA molecules,

registration of a spectrum of circular dichroism (abnormal opticalactivity) in the region of BAS absorption,

determination of the BAS presence by the shape of a band in the spectrumof circular dichroism.

However, the exact determination of BAS concentration and, consequently,the practical opportunities of the specified method for BASdetermination are limited by the following factors:

difficulty of creation of films (gels) that are adequate to the certainphysico-chemical requirements (neutral in relation to DNA character of afilm, its transparency, optical isotropy, etc.),

difficulty in the maintenance of constant properties of DNA liquidcrystals in a film structure, even during time for diffusion of BAS,

significant interval of time, after which the registration becomespossible of an appreciable value of the optical signal arising as aresult of diffusion of BAS molecules into a polymeric film and theirsubsequent interaction with nucleic acid moleculs forming the liquidcrystalline dispersion,

impossibility for exact determination of the value of the abnormaloptical signal in the UV-region of the spectrum (i.e., in the DNAabsorption region), that is caused by an unsufficient transparency offilms (gels) in this part of the spectrum. Therefore, though thepresence of the substance under analisis can be registered, the exactdetermination of its concentration is extremely difficult,

application for registration of the abnormal optical signal of theexpensive stationary dichrographs (Jobin-Yvon, Mark III or Mark V;Jasco, Model 710/720) being available, as a rule, only in specializedscientific laboratories. A lack of these devices is not only their highcost but also low speed of registration of the optical signal.

A well known dichrograph of “Jasco” firm (Model J-710/720Spectropolarimeter, Instruction Manual: Jasco Corporation, 2967-5,Ishikwa-Cho, Hachioji City, Tokyo, Japan (June, 1990)), which can beapplied as a device for determination of BAS in an analyzed liquid,comprises installed consistently:

a source of light radiation,

a selector forming a light flow of a certain wavelength;

a polarizer forming a linearly polarized light of the specified lightflow;

a modulator of polarization transforming the linearly polarized lightflow into a circularly polarized light flow with a periodically varieddirection of rotation of its polarization vector;

a cell for an analyzed sample;

a photodetector transforming an optical signal generated by componentsof the sample to be analyzed into the proportional electrical signal;

a synchronous amplifier of the specified electrical signal;

a processing unit for processing the received electrical signal and forcalculation of the biologically active substance concentration;

a control module.

The specified dichrograph comprises as a selector a low lighttransmittance double-prism monochromator based on synchronous rotationof two half-prisms by means of an electromechanical driver containing alever mechanism actuated with an electric motor. The light from a sourceof light radiation passes trough the double-prism monochromator, thosetwo prisms are tuned on the certain wavelength. The presence of thelever mechanism enables to tune the specified monochromator on differentwavelengths of the light flow leaving the monochromator.

However, because of high inertia of the specified driver containing thelever mechanism, the specified selector has a small speed of wavelengthtuning. As a result, the usage of the specified dichrograph for BASdetermination significantly increases the time of the sample analysis.This factor, at the analyses of biological liquids such as the blood,the urea, etc., can be accompanied by a damage in the health, and insome cases—in the life of patients.

Moreover, because of complex design, the monochromator has large losseson its optical elements, therefore it has a low light transmittance thatresults in the deterioration of sensitivity of the device as a whole anddoes not permit to determine low BAS concentrations in analyzed liquids.In addition, the specified device is multifunctional as well, has largedimensions and weight, that requires a specially equipped room for itsinstallation and exploitation. As the result, the specified device has alow mobility and cannot be used for the urgent analyses in cliniclaboratories or directly in hospital wards.

The factors named above make fast obtaining the information about theBAS concentration in analyzed liquids difficult and limit wideapplication of optical systems for such kind of analyses in conditionsof clinics and laboratories.

BRIEF SUMMARY OF THE INVENTION

The problem is used as a basis for the offered invention to create amethod of determination of BAS in an analyzed liquid with suchconditions of its realization and such a device that would allow fastlyand precisely to determine the concentrations (low, in particular) of abiologically active substance capable to interact with lineardouble-stranded DNA molecules in any liquids including biological ones,such as blood plasma, full blood, etc.

This problem is solved by the creation of the method for determinationin an analyzed liquid of a biologically active substance interactingwith a cholesteric lyotropic liquid-crystalline DNA dispersion formed ina polymer neutral in relation to the DNA, and in this method, accordingto the invention, the cholesteric lyotropic liquid-crystallinedispersion is formed of the linear double-stranded DNA molecules of lowmolecular mass immediately before its mixing with an analyzed liquidcontaining the determined biologically active substance, in additionthis analyzed liquid is mixed previously with the specified polymerunder conditions, at which optical properties of the lyotropicliquid-crystalline DNA dispersion are not broken, then through theanalysed sample obtained as the result of the indicated mixing of theprepared analyzed liquid with the specified liquid-crystalline DNAdispersion, a flow of circularly polarized light is passed, and theoptical signal generated by the liquid-crystalline dispersion isregistered at two wavelengths, one of them is in the region of the DNAnitrogen bases absorption, and another one is in the region ofabsorption of the determined biologically active substance, after this aratio between these signals at the specified wavelengths is calculated,and the concentration of the biologically active substance is determinedon this ratio magnitude using the calibration curve.

DETAILED DESCRIPTION OF THE INVENTION

The theory of forming DNA liquid-crystalline dispersions in polimersolutions neutral in relation to this macromolecule is described in[Yevdokimov Yu. M., Skuridin S. G., Salyanov V. I., 1988, LiquidCrystals, 3, p.p.1443-1459], and experimental “boundary” conditions, atwhich the abnormal optical activity of DNA liquid-crystallinedispersions is preserved, are done in [Evdokimov Yu. M., Skuridin S. G.,Akimenko N. M., 1984, Russian Journal: Vysokomolekulyarnye coedineniy,A24, p.2403-2410]. Use of the offered method allows quickly, simply,precisely and with a high accuracy and sensitivity to determine anybiologically active substances capable to interact with lineardouble-stranded DNA molecules in any liquids where it is possible tocreate the conditions for conservation of the liquid-crystalline DNAdispersions; in addition, it is possible to carry out the analysis inany laboratories where the specially organized rooms and the specialqualification of the technicians are not required.

The offered way is especially important for fast, precise and highsensitive determination of the presence and the concentration (includinglow) of biologically active substances (antitumor compounds,antibiotics, proteins, etc.) in blood of patients in the practice ofoncology, surgery, gynecology, at medico-ecological screening, and ithelps to rescue of health and life of patients when other methods areinapplicable or do not give the proper results. It is expedient as aneutral polymer to use the polyethyleneglycol, because this polymer isharmless for an experimentator, chemically neutral in relation to DNA,has a high solubility necessary for creation of conditions of the DNAphase exclusion, the optical isotropy and high transparency necessaryfor measurement of spectra of circular dichroism; moreover, differentmolecular mass preparations of this polymer of the reasonable price areavailable.

It is desirable as an analyzed liquid to use the biological liquidbecause the pharmaco-kinetics of biologically active substances is aimedfor application in such liquids as blood, urea, etc.

It is favorable as a biological liquid to use the plasma of bloodbecause a number of the factors influencing accuracy of determination ofa biologically active substance decreases in this case.

It is expedient that the biologically active substance should representan antitumor compound of the anthraquinon group because these compoundsare widely used separately as well as in various combinations forchemotherapy of oncological diseases.

It is desirable that the antitumor compound of the anthraquinone groupwould represent the mithoxantrone because the mithoxantrone is one ofthe most powerful antitumor agent of a wide spectrum of action.

It is favorable that at determination of the biologically activesubstance in a biological liquid with heterogeneous in it distributionof the biologically active substance its concentration obtained usingthe calibration curve should be corrected in view of the coefficient ofits distribution between components in a biological liquid.

Thus, the offered method can be used for the fast, precision and highsensitive determination of the presence and the concentration of variousbiologically active substances (antitumor preparations, antibiotics,proteins, etc.) in various liquids including the blood of patients inthe practice of oncology, surgery, gynecology, at medico-ecologicalscreening and at the help to rescue of health and life of patients whenother techniques are inapplicable or do not give the proper effect.

The problem presented is solved also by creation of the device fordetermination of a biologically active substance in an analyzed liquid,comprising installed consistently: a source of light radiation; aselector having at least one optical element and forming a light flow ofa certain wavelength; a polarizer forming a linearly polarized light ofthe pointed light flow; a modulator of polarization transforming thelinearly polarized light into a circularly polarized light flow with aperiodic change of the direction of rotation of its polarization vector;a cell for the analized sample; a photodetector transforming the opticalsignal generated by components of the sample into the proportionalelectrical signal; a synchronous amplifier increasing the pointedelectrical signal; a processing unit for processing the obtainedelectrical signal and for calculation of the biologically activesubstance concentration; a control module, and in this device, accordingto the invention, the selector contains an electrodynamic driver of apositional type, designed with a possibility of setting at least twowavelengths, at which the optical signal generated by the components ofthe sample under analysis is registered, and at least one opticalelement fixed on a motor shaft of the specified driver.

The presence of the specified driver and the optical element fixed onits motor shaft allows quickly and precisely to set the requiredwavelengths and to tune the device from one wavelength to another one,that allows to reduce a time period for the selection of the requiredwavelength by ten, at least, with simultaneous decreasing an electricpower consumption; it allows quickly, precisely and with a highsensitivity to determine the concentration (including low) ofbiologically active substances capable to interact with thedouble-stranded DNA molecules in the analyzed liquids including thebiological liquids, such as full blood, plasma of blood, etc.

It is expedient that the selector should represent a simplemonochromator containing at least two optical elements, one of themrepresents a dispersive element, and one of these optical elements isfixed on the motor shaft of the specified driver with the possibility toturn around its own axis. In addition the dispersive element canrepresent a diffraction grating that can be designed concave.

It is possible that the selector should represent a simple monochromatorcontaining one optical element fixed on the motor shaft of the specifieddriver with the possibility to turn around its own axis, and thisoptical element must be the dispersive element designed as the concavediffraction grating.

The presence of the simple monochromator with minimal number of opticalelements simplify a design of the device as a whole that allowsconsiderably to reduce its dimensions. It results in significantcheapening of the device and allows to equip with this device anylaboratories, as the offered device does not require a presence ofspecially organized place and special qualification of the personel.Moreover, the usage of the simple monochromator allows to simplify theoptical system of the device, to reduce losses of light on opticalelements, that is to increase a light flow transmittance, and, due tothis, to increase the sensitivity of the device, to reduce its size and,hence, to ensure the mobility of the device for its use under anyconditions. Use of the diffraction grating as the dispersive elementgives a possibility to keep the required resolving force of themonochromator, so not to lose in the spectral resolution of theselector.

It is possible that the selector should contain a grate number ofoptical elements being a set of narrow-band interference filters fixedon the motor shaft of the specified driver with a possibility of theiralternate introduction into the light flow, in addition each of thesefilters has a transparency band in the region of the certain wavelengthchosen for the concrete biologically active substance.

The usage in the selector of a set of the interference filters fixed onthe motor shaft of the electrodynamic driver of the positional typeallows, at maintenance of a high speed of the selection of the requiredwavelength, essentially to simplify the optical scheme of the selectorand, hence, to increase the transmittance of the light flow and toreduce the dimensions and the weight of the whole device.

It is expedient that the electrodynamic (galvanometric) driver shouldcontain a motor having both a stator and a rotor, and the rotor turnangle transducer, representing an inductive differential converter ofthe rotor turn angle into the electrical signal, and containing amodulator that is designed as a ring fixed on the motor shaft with aneccentricity concerning its rotation axis.

Due to the presence of the specified eccentricity there is a possibilityto realize the required character of the dependence of the signalamplitude of the turn angle transducer, i.e. the wavelength of theselector, upon the turn angle of the motor shaft, and the character ofthis dependence is determined by the form (design) of the modulator.

Thus, the usage of the offered method and the offered device allowsquickly, precisely and with a high sensitivity to determine the presenceand the concentration of various biologically active substances(antitumor compounds, antibiotics, proteins, etc.) in various liquidsincluding biological liquids, for example, in blood of patients, inpractice of oncology, surgery, gynecology, at

the abnormal optical activity of the sample is measured at twowavelengths, one of them is in the region of absorption of DNA nitrogenbases, and another one is in the region of BAS absorption. (The band inthe region of the DNA absorption is used as the internal standard forthe “quality” of the liquid-crystalline dispersions formed, and alsoreflects the stability and the correctness of action of the offereddevice; the band in the spectrum of circular dichroism in the region ofBAS absorption represents by itself as the indicator of the BAS presencein the analysed sample, and the amplitude of this band reflects theconcentration of the BAS),

the ratio is calculated between the values of the optical signals atwavelengths indicated above,

the exact BAS concentration is determined due to the calibration curvethat reflects the dependence of values of the signals ratio upon the BASconcentrations and is previously constructed according to the methoddescribed above.

For the best understanding of the present invention, a few examplesdescribing various stages of the declared method, with the references tothe applied drawings is given below. Here:

FIG. 1 characterizes the spectrum of absorption of the initialaqueous-salt solution of the linear double-stranded DNA used forformation of particles of the cholesteric liquid-crystallinedispersions, in coordinates: “optical density, A”—“λ, wavelength”;

C_(DNA)˜80 μg/ml;

Chicken erythrocyte DNA (“Reanal”, Hungary);

Mol.mass of DNA˜(3-5)×10⁵ Da.

FIG. 2 characterizes the circular dichroism spectra of the initial DNAand its cholesteric liquid-crystalline dispersion in coordinates:“Δε=ε_(L)−ε_(R)”—“λ, wavelength”, where:

value of Δε=ΔA/C_(DNA)—the molar circular dichroism;

ε_(L)—the dichroism for the left-circular-polarized light; presence inthe analysed sample, and the amplitude of this band reflects theconcentration of the BAS),

the ratio is calculated between the values of the optical signals atwavelengths indicated above,

the exact BAS concentration is determined due to the calibration curvethat reflects the dependence of values of the signals ratio upon the BASconcentrations and is previously constructed according to the methoddescribed above.

BRIEF DESCRIPTION OF DRAWINGS

For the best understanding of the present invention, a few examplesdescribing various stages of the declared method, with the references tothe applied drawings is given below. Here:

FIG. 1 characterizes the spectrum of absorption of the initialaqueous-salt solution of the linear double-stranded DNA used forformation of particles of the cholesteric liquid-crystallinedispersions, in coordinates: “optical density, A”—“λ-wavelength”;C_(DNA)˜80 μg/ml; Chicken erythrocyte DNA (“Reanal”, Hungary); Mol.massof DNA˜(3-5)×10⁵ Da.

FIG. 2 characterizes the circular dichroism spectra of the initial DNAand its cholesteric liquid-crystalline dispersion in coordinates:“Δε=ε_(L)−ε_(R)”—“λ-wavelength”, where:

value of Δε=ΔA/C_(DNA)—the molar circular dichroism;

ε_(L)—the dichroism for the left-circular-polarized light;

ε_(R)—the dichroism for the right-circular-polarized light;

ΔA—the experimentally measured circular dichroism,

C_(DNA)—the DNA concentration;

the curve 1—the CD spectrum of the cholesteric liquid-crystalline DNAdispersion;

the curve 2—the CD spectrum of the aqueous-salt solution of the initiallinear DNA;

The curve 1 (right ordinate): C_(PEG)=170 mg/ml, PEG mol. mass=4000(“Ferak”, Germany); 0.3 M NaCl+10⁻² M phosphate buffer; pH˜7.0.

The curve 2 (left ordinate): 0.3 M NaCl+10⁻² M phosphate buffer; pH˜7.0.

FIG. 3 characterizes the absorption spectrum of the mithoxantrone usedas an example of BAS, in coordinates: “optical density, circulardichroism A”—“wavelength, λ”;

0.3 M NaCl+10⁻² Ml phosphate buffer; pH˜7.0; C_(t MX)=1.5×10⁻⁵ M.

FIG. 4 characterizes the circular dichroism spectra of theliquid-crystalline DNA dispersion treated with different concentrationof the mithoxantrone, in coordinates: “circular dichroismΔA=A_(L)-A_(R)”—“wavelength, λ”, where:

A_(L)—the dichroism for the left-circularly polarized light;

A_(R)—the dichroism for the right-circularly polarized light;

ΔA—the experimentally measured circular dichroism,

1-C_(t MX)=0; 2-C_(t MX)=1.55×10−6 M;

3-C_(t MX)=3.08×10⁻⁶ M; 4-C_(t MX)=5.35×10⁻⁶ M;

C_(DNA)=20 μg/ml; C_(PEG =)170 mg/ml;

0.3 M NaCl+10⁻² M phosphate buffer; pH˜7.0;

“ΔA=A_(L)-A_(R)” is in mm; 1 mm=5×10⁻⁵optical units; L=1 cm.

FIG. 5 reflects the dependence of the amplitude (ΔA₆₈₀) of the negativeband at λ=680 nm in the circular dichroism spectrum of theliquid-crystalline DNA dispersion upon the concentration (C_(t MX)) ofthe mithoxantrone added; the arrow in the Fig. designates themithoxantrone concentration, down to which between values of ΔA₆₈₀ andC_(t MX) directly proportional dependence is observed;

C_(DNA)=20 μg/ml; C_(PEG)=170 mg/ml;

0.3 M NaCl+10⁻² M phosphate buffer; pH˜7.0

ΔA₆₈₀ is in mm; 1 mm=1×10⁻⁶ optical units; L=1 cm.

FIG. 6 shows the dependence of the amplitude of the band in the circulardichroism spectra of the liquid-crystalline dispersions of the (DNA-MX)complexes at λ=680 nm upon the value C_(t MX) in the analyzed samplesand in the control solutions (1 and 2, respectively). In FIG. 6(A→B→C=C_(t Mx)) the way for determination of C_(t MX) in the analyzedsample is shown that takes into account the binding of the mithoxantronewith form elements of blood and high-molecular components of plasma ofdifferent chemical nature;

C_(DNA)=20 μg/ml; C_(PEG)=170 mg/ml;

0.225 M NaCl+7.5×10⁻³ M phosphate buffer; pH˜7.0.

ΔA₆₈₀ is in mm; 1 mm=2×10⁻⁶ optical units; L=1 cm.

FIG. 7 reflects the dependence of the C_(t MX) ^(sample) magnitude onthe C_(tMX) ^(control) value;

C_(DNA)=20 μg/ml; C_(PEG)=170 mg/ml;

0.225 M NaCl+7.5×10⁻³ M phosphate buffer; pH˜7.0.

FIG. 8. shows the dependence of the ratio of the bands in the CD spectraof the liquid-crystalline dispersions of the (DNA-MX) complexes atλ₁=680 nm and λ₂=275 nm upon the C_(t Mx) ^(sample) value. In FIG. 8 thedifferent points concern to experiments carried out with the use ofblood of four different donors;

C_(DNA)=20 μg/ml; C_(PEG)=170 mg/ml;

0.225 M NaCl+7.5×10⁻³ M phosphate buffer; pH˜7.0.

ΔA₆₈₀ and ΔA₂₇₅ are in mm; 1 mm=2×10⁻⁶ optical units; L=1 cm.

FIG. 9. represents the basic scheme of stages for preparation of ananalysed sample of blood for determination of the presence of a BASinteracting with the liquid-crystallinr DNA dispersion. (It is necessaryto pay attention on the fact resulted from the offered method of bloodpreparation that the initial BAS concentration decreases by four).

FIG. 10 represents the block diagram of the device designed according tothe invention;

FIG. 11 schematically shows the selector representing a simplemonochromator designed according to the invention, top view;

FIG. 12 schematically represents the electrodynamic driver of thepositional type designed according to the invention, top view with thecomplex section;

FIG. 13 schematically shows the selector representing a set ofinterference filters that is designed according to the invention,isometry;

FIG. 14—the CD spectrum of the aqueous-salt solution of n-propylammoniumsalt of d-10-camphorsulphonic acid registered by the offered device;

C₁₀H₁₆O₄S concentration 0.15 mg/ml;

wavelength scan step=5 nm/div.

FIG. 15—the CD spectrum of the aqueous-salt solution of the linearB-form DNA registered by the offered device;

C_(DNA)=5 μg/ml; 0.3 M NaCl+10⁻² M phosphate buffer; pH˜7.0;

wavelength scan step=3.5 nm/div.

FIG. 16—the CD spectrum of the liquid-crystalline DNA dispersionregistered by the portable dichrometer;

C_(DNA)=5 μg/ml; C_(PEG)=170 mg/ml;

0.3 M NaCl+10⁻² M phosphate buffer; pH˜7.0;

wavelength scan step=10 nm/div.

FIG. 17—the CD spectrum of the liquid-crystalline dispersion of thecomplex (DNA-MX) registered with help of the portable dichrometer;

C_(DNA)=5 μg/ml; C_(PEG)=170 mg/ml;

0.3 M NaCl+10⁻² M phosphate buffer; pH˜7.0;

C_(t MX)=8.623×10⁻⁷ M.

wavelength scan step=7 nm/div.

FIG. 18—the dependence of the optical signal generated by theliquid-crystalline DNA (λ=680 nm) upon the total concentration of MX.The various black points designate the data received by the portabledichrometer in different series that are distinguished by MXconcentration.

EXAMPLE 1 Forming the Liquid-Crystalline DNA Dispersion

1.1. Probes of NaCl (17.532 g), NaH₂PO₄×2 H₂O (0.78 g) and Na₂HPO₄×12H₂O(1.79 g) are placed into a calibrated flask (V =1000 ml) and dissolvedin distillated water; water is added up to a label.

By such a way 1 L of 0.3 M of NaCl solution containing 10⁻² M ofphosphate buffer is prepared.

1.2. 10 mg of the double-stranded DNA preparation (“Reanal”, Hungary;molecular mass (0.3-0.5)×10⁵ Da) are placed into a calibrated flask(V=10 ml) and dissolved in the solution prepared according to item 1.1.The volume of the solution is added up to a label.

By such a way 10 ml of aqueous-salt solution of the DNA with its fixedconcentration are prepared. The DNA concentration is determined using aspectrophotometer and being derived from the ratio: 1 mg of the DNAcorresponds to 20 optical units in 1 ml (λ_(max)=258.4 nm; pH˜7.0).

1.3. Probes of NaH₂PO₄×2H₂O (0.078 g) and Na₂HPO₄×12H₂O (0.179 g), NaCl(1.7532 g) and polyethyleneglycol (PEG) (“Ferak”, Germany; PEG molecularmass 4000; 34 g) are placed into a calibrated flask (V=100 ml) anddissolved in distillated water; water is added up to a label.

By such a way 100 ml of aqueous-salt solution of PEG (C_(PEG)=340 mg/ml;0.3 M NaCl+10⁻² M phosphate buffer) are prepared.

1.4. 4.6 ml of the solution 1.1 are mixed with 0.4 ml of the solution1.2 in a glass test-tube (V=10 ml).

By such a way 5 ml of aqueous-salt solution of the double-strandedlinear DNA are prepared (C_(DNA)=80 μg/ml; 0.3 M NaCl+10⁻² M phosphatebuffer).

After mixing solutions 1.1 and 1.2 the absorption spectrum is registeredof aqueous-salt solution of the given DNA concentration preparedaccording to item 1.4 (FIG. 1, where the absorption spectrum of the DNAaqueous-salt solution is represented, C_(DNA)˜80 mkg/ml; DNA of chickenerythrocytes (“Reanal”, Hungary); DNA mol. Mass˜(3-5)×10⁵ Da).

1.5. 4 ml of the solution 1.3 are mixed with 4 ml of the solution 1.4 ina glass test-tube (V=15 ml); the mixture obtained is intensively mixedduring 3 min.

By such a way 8 ml of the liquid-crystal DNA dispersion (C_(DNA)=40μg/ml; C_(PEG)=170 mg/ml; 0.3 M NaCl+10⁻² M phosphate buffer).

After mixing, the circular dichroism (CD) spectrum is registered of themixture prepared on item 1.5. The intense negative band in the region ofabsorption of the DNA nitrogen bases (FIG. 2, curve 1) testifies that,as a result of mixing solutions prepared on items 1.3 and 1.4, theliquid-crystalline DNA dispersion of the cholesteric type is formed.

EXAMPLE 2 Optical Properties of the Liquid-Crystalline DNA DispersionTreated with Mithoxantrone

2.1. A probe 0.4 mg of mithoxantrone (MX; the antitumor substance of theanthraquinone group) is placed into a test-tube (V =0.5 ml) anddissolved in 200 μl of the solution 1.1.

By such a way 200 μl of the MX aqueous-salt solution (C_(MX)=2 mg/ml;0.3 M NaCl+10⁻² M phosphate buffer) are prepared.

2.2. The MX concentration in the solution 2.1 is determined byspecrophotometer (“Specord” M 40, Germany), using the known meaning ofMX molar extinction coefficient (ε=21500 M⁻¹ cm⁻¹; λ_(max)=660 nm). Themolar MX concentration in the solution 2.2 is 3.868×10⁻³ M.

In FIG. 3 the absorption spectrum of the MX aqueous-salt solution isshown.

2.2. 2 ml of the standard liquid-crystalline DNA dispersion prepared onitem 1.5 are placed into a rectangular optical quartz cell (V=4 ml;optical length=1 cm), and its CD spectrum is registered in thewavelength region 750-220 nm by a dichrograph “Jobin-Yvon, Mark III”(France).

2.3. 1 μl portions of the solution 2.1 (total volume of the addedsolution 2.1 is 6 μl) are put into the cell containing 2 ml of the DNAdispersion prepared on item 2.2; after each portion of the solution 2.1,the solution in the optical cell is mixed, and the CD spectrum isregistered in the wavelength region 750-220 nm.

In FIG. 4 the CD spectrum of the initial liquid-crystalline DNAdispersion (the curve 1) is compared to that of the same dispersionadded with different MX concentrations C_(t MX) (C_(t MX)—the totalconcentration of MX added in the solution of the liquid-crystalline DNAdispersion). The MX addition is accompanied by an appearance of anadditional intense negative band in the region of MX absorption (680 nm)in the CD spectrum of the liquid-crystalline DNA dispersion. Inaddition, the amplitude of the band at the wavelength λ=680 nm “isconnected” to the amplitude of the band at λ=275 nm in a following way:at identical concentration of MX if the amplitude of the 680 nm band islarger, then the amplitude of the λ=275 nm band is higher, and on thecontrary.

The dependence of the amplitude of the band at λ=680 nm in the CDspectrum of the liquid-crystalline dispersion of complexes (DNA-MX) uponCt MX (FIG. 5) shows that between the value of AA at 680 nm and C_(t MX)directly proportional dependence is observed in the range of MXconcentrations from 0 down to 12×10⁻⁶ M . As the amplitude of the bandin the CD spectrum in the region of MX absorption (ΔA₆₈₀) depends onlyon the concentration of MX molecules bound (C_(b MX)) in a complex withthe DNA molecules, and as between values of C_(t MX) and C_(b MX) thereis a simple ratio:

C _(t MX) =C _(b MX) +C _(free MX)  (1)

where: C_(free MX)—concentration in the solution of free (unbound in acomplex with the DNA) MX molecules, one can consider directlyproportional dependence between values ΔA₆₈₀ and C_(t MX) as theindication to a low concentration of free MX in the solution.

The value of C_(b MX), at the fixed DNA concentration, is closer to theC_(t MX) value, then the last concentration is lower. In the area of thepharmaceutically relevant meanings of MX concentrations (ng/ml), one canaccept that the value of C_(b MX) is approximately equal to the value ofC_(t MX).

Thus, the intense band in the region of MX absorption in the CD spectrumin combination with the presence of the directly proportional dependenceof the amplitude of this band on the concentration of MX added can beused for determination of the MX presence and concentration.

At determination of the concentration of MX molecules that can interactwith DNA molecules, and, hence, of the MX concentration in biologicalliquids by the circular dichroism spectra, it is necessary to take intoaccount that this determined value could be influenced by two factors:

1) Distribution of MX molecules between form elements of blood,

2) “Heterogeneity” of optical properties of the liquid-crystalline DNAdispersions in blood of different patients, influencing the amplitude ofthe band at λ=275 nm, and, hence, the amplitude of the band at λ=680 nm.

Therefore for the accurate determination of the MX concentration it isnecessary to take into account the influence of these factors.

EXAMPLE 3 Consideration of Mithoxantrone Distribution in Blood ofPatients

At determination of MX concentration in biological liquids (blood, bloodplasma, etc.) it is necessary to take into account a possible binging ofMX coming in blood with form elements of blood and different originhigh-molecular components of blood plasma. An example below allows toestimate a degree of MX binding.

3.1. Blood probes (in 2 ml) are spilled into 8 centrifuge polypropylenetest-tubes (V=12.5 ml).

3.2. 0, 1, 3, 5, 7, 9, 11 and 13 μl of the solution prepared on item2.1. are added correspondently in each of test-tubes (item 3.1).

3.3. After addition of the solution 2.1 on the blood probes each of theprepared probes is mixed.

By such a way a series of MX-containing samples of blood in whichC_(t MX) varies from 0 down to 24.98×10⁻⁶ M is prepared.

3.4. In 2 ml of the solution prepared on item 1.3 are added on each ofMX-containing blood samples prepared according to items 3.1-3.3.

3.5. After addition of the solution 1.3 on blood samples (item 3.3) theprepared solutions are mixed.

By such a way MX-containing blood samles in the PEG aqueous-saltsolution (C_(t MX) from 0 down to 12.49×10⁻⁶ M; C_(PEG)=170 mg/ml; 0.15M NaCl+5×10⁻³ M phosphate buffer) are prepared.

3.6. The blood samples prepared according to item 3.5 are certrifugated(12,000 rev/min; 4° C.; centrifuge K24D, Germany).

As a result of centrifugation, a deposit consisting of form elements ofblood and high-molecular components of blood plasma is formed.

3.7. After low speed centrifugation in 2 ml of the supernatant from eachof centrifuge test-tubes (item 3.6; 8 test-tubes) are selected andtransfered into 8 corresponding glass test-tubes (V=10 ml).

3.8. In 2 ml of the standard liquid-crystalline DNA dispersion formedaccording to item 1.5 are added in each of 8 glass test-tubes containingin 2 ml of the supernatant (item 3.7).

3.9. The solutions prepared according to item 3.8 are stirred.

By such a way a series of the analyzed samples is prepared that containthe initial liquid-crystalline DNA dispersion and the supernatant ofPEG-containing samples of blood with different MX contents (C_(DNA)=20μg/ml; C_(PEG)=170 mg/ml; 0.15 M NaCl+5×10⁻³ M phosphate buffer).

3.10. In parallel with the sample series (item 3.9), a control series ofsamples according to operations on items 3.1-3.9 is prepared, where,instead of blood, the solution 1.1 is used. (In the control seriesC_(t MX) varies from 0 down to 6.245×10⁻⁶ M).

3.11. After mixing the analyzed samples (item 3.9) and the controlsolutions (item 3.10) the CD spectra of the analyzed (item 3.9) and thecontrol (item 3.10) sample series are registered with a dichrograph“Jobin-Yvon, Mark III” (France).

The amplitude of the band in the CD spectra (λ=680 nm) of PEG containingsamples of blood (item 3.4) grows in accordance with increasing C_(t MX)(FIG. 6, curve 1).

Distinction between lines 1 and 2 (FIG. 6) shows that the analyzed bloodsamples contain smaller, in comparison with the control samples,concentration of MX. The reduction of MX concentration reflects the factthat the part of MX molecules binds to form elements of blood andhigh-molecular components of plasma and, hence, becomes inaccessible(item 3.6) for determination by means of the optical method.

For consideration of the divergence between the same concentrationmeanings (C_(t MX)) of MX added in the blood and in the controlsolutions, a technique shown in FIG. 6 is used.

According to this technique the amplitude of the band, that ischaracteristic for the CD spectrum of the analyzed blood sample at λ=680nm (line 1, point “A”), is transferred on line 2 (point “B”) thatrepresents the dependence of the value of ΔA₆₈₀ upon C_(t MX) for thecontrol series of solutions: A → B → C = C_(tMX)^(sample).

The meaning of the value ΔA₆₈₀ in a point “B” on the absciss axis iscorresponded to the value of C_(t MX) (point “C”), i.e. the meaning ofthe MX concentration in the analyzed sample (C_(t MX) ^(sample))

In FIG. 7 the dependence of the MX concentration in the analyzed bloodsamples (C_(tMX) ^(sample)) upon its value in the control solutions(C_(t MX) ^(control)) is presented.

The dependence between these values is described by the straight linewith the inclination angle tangent 0.384. It means that under conditionsused only 38.4% of total MX molecules added in blood samples remainaccessible to the analysis by the optical methods. This value should beentered into the final equation for determination of the concentrationof MX added in blood.

EXAMPLE 4 Consideration of Probable “Heterogeneity” of OpticalProperties of the Liquid-Crystalline DNA Dispersions at Calculation ofMX Concentration in an Analyzed Biological Liquid

4.1. To exclude the “heterogeneity” of optical properties of theliquid-crystalline DNA dispersion in blood of different patients, and,hence, the value of the optical signal generated by theseliquid-crystalline dispersions at formation of the (DNA-MX) complex, themeanings of ΔA₆₈₀ received in experiments on determination of MX werenormalized on the value of the optical signal registered in the CDspectrum of the control liquid-crystalline DNA dispersion at λ=275 nm(ΔA₂₇₅).

4.2. Dependence of the ratio (ΔA₆₈₀/ΔA₂₇₅) upon the value C_(t MX)^(sample) (FIG. 8) received for blood of four different donorsrepresents an universal calibration curve, and using this curve it ispossible to determine values of C_(t MX) ^(sample) in the region of MXconcentrations from 0 down to 2.5×10⁻⁶ M.

EXAMPLE 5 The Algorithm for Determination of Concentration of aBiogically Active Substance in Blood of a Patient

The preparation of blood taken from a patient and containing abiologically active substance, and the estimation of BAS concentrationis carried out according to the scheme shown in FIG. 9. In accordancewith this scheme tested on the example of determination of MXconcentration:

5.1. 4 ml of blood taken from a patient are mixed In polypropylenecentrifuge test-tube (V=12.5 ml) with 4 ml of the solution on item 1.3.

5.2. The prepared mixture is stirred.

By such a way the blood sample is prepared that contains a biologicallyactive substance (BAS) in the PEG aqueous-salt solution.

5.3. The BAS-containing blood sample in the PEG aqueous-salt solutionprepared on item 5.2 is centrifugeted.

5.4. After low-speed centrifugation, 5 ml of the supernatant are mixedwith 5 ml of the standard liquid-crystalline DNA dispersion prepared onitems 1.1-1.5 (example 1).

By such a way the analyzed blood sample is prepared.

5.5. In parallel with preparation of the blood sample, the controlliquid-crystalline DNA dispersion is prepared on items 5.1-5.4 where,instead of blood, the solution on item 1.1 is used.

5.6. After mixing the analyzed blood sample (item 5.4) and the controlliquid-crystalline DNA dispersion (item 5.5), the amplitudes of thebands in the CD spectra are registered, accordingly, at the wavelengthλ₁ in the region of BAS absorption (in our case—at 680 nm; ΔA₆₈₀) and atthe wavelength λ₂ that is appropriate to the region of DNAabsorption(275 nm; ΔA₂₇₅).

5.7. The ratio ΔA₆₈₀/ΔA₂₇₅ is calculated.

5.8. The received ratio (item 5.7) is put on the universal calibrationcurve (FIG. 8, example 4.2), and the meaning of C_(t BAS(MX)) ^(sample)value that corresponds to this ratio is determined.

5.9. After determing the value of C_(t BAS(MX)) ^(sample) and takinginto account 4-time dilution of the blood sample on MX concentration viathe expression:

C ⁰ _(t BAS (MX))=4×C _(tBAS (MX)) ^(sample)  (2)

the concentration C_(t BAS(MX)) of BAS (MX) nonbound with form elementsof blood is determined in blood of a patient.

5.10. For determination of the initial concentration of BAS (MX) theamendment on item 3.11 is entered:

C⁰ _(t BAS (MX))—38.4%

C_(t BAS (MX)) ^(initial)—100%

C _(t BAS (MX)) ^(initial) =C _(tBAS (MX)) ^(sample)×100/38.4=4×2.6×C ⁰_(t BAS (MX))  (3)

The minimum concentration of BAS (MX), that is determined in blood of apatient by means of the offered technique based on the measurement ofthe optical signal generated by the liquid-crystalline DNA dispersionwith a dichrograph “Jobin-Yvon, Mark III” (France), makes 5×10⁻⁷ M.

BEST MODE OF CARRYING OUT THE INVENTION

Below a specific example of fulfilment of the offered device forrealization of the offered method with the references to the appliedfigures is given.

The device for the determination of BAS in an analyzed liquid designedaccording to the invention comprises: the source 1 (FIG. 10) of lightradiation made, for example, on the base of the xenon lamp with aircooling; the selector 2 of light radiation wavelengths forming a lightflow in a certain narrow spectral interval of wavelengths; the polarizer3 designed, for example, as a prism of a nonlinear crystal material,fixed after the selector 2 and forming a linearly polarized light flowof the specified one; the modulator 4 of polarization, for example, of aphotoelastic type, made of quartz, placed behind the polarizer 3 andtransforming the linearly polarized light flow into a circular-polarizedlight flow with a periodically varied direction of rotation of itspolarization vector; the cell 5 for placing of the analysed sample whichcontains a mixture prepared according to the method described above ofthe analyzed liquid containing BAS and the lyotropic cholestericliquid-crystalline DNA dispersion; the photodetector 6 with thephotosensitive surface that is oriented in the direction of thespecified cell 5 and that transforms the optical signal generated by thespecified liquid-crystalline dispersion into a proportional electricalsignal. The photodetector 6 has two outputs 7, 8, one 7 of which isconnected to an entry of control of the power supply 9 of thephotodetector 6, and another one 8 is connected to the first entry 10 ofthe synchronous amplifier 11, output 12 of which is connected to anentry 13 of the unit 14 for processing the received electrical signaland for calculation of the concentration of the biologically activesubstance, other entry 15 of the specified processing unit is connectedto the first output 16 of the control module 17, the second output 18 ofthe control module 17 is connected to the modulator 14 of polarization,the third output 19 of the control module 17 is connected to theselector 2, and the fourth its output 20 is connected to the secondentry 21 of the synchronous amplifier 11.

As the unit 14 for processing the received electrical signal andcalculation of the concentration of BAS, a personal computer, forexample, or any other means destined for the similar purposes can beused.

The selector 2 forming the light flow of the certain wavelength can havevarious constructive designs, the common advantages of them are aminimum number of optical elements that is necessary for receiving themaximum output light flow at conservation of the required resolution andmeasurement accuracy (a high light flow transmittance), and the presencein the selector 2 of the electrodynamic driver 22 of the positional typeensuring a turn of one (or several) optical elements with a possibilityof setting two chosen for the determined BAS wavelengths at which the CDsignal measuring is produced (for example, a known electrodynamic driveris described in [Nesteruk I. N., Kompanets O. N., Mishin V. I., RussianJournal: Kwantovaya Elektronika, 1988, 15, N3, p.p.455-459].

In FIG. 11 the selector 2 is shown representing the simple wavelengthtuned monochromator 23 comprising fixed on the general basis 24 theentry slit 25 and three optical elements 26, 27, 28. One of the opticalelements represents the collimating mirror 26, the second opticalelement is the focusing mirror 27, and the third optical element is thedispersive element 28, designed as the plane diffraction grating. Theconcave surface 29 of the collimating mirror 26 is oriented in thedirection of the entry slit 25 and the working surface 30 of thedispersive element 28. The concave surface 31 of the focusing mirror 27is oriented simulteneously in the direction of the working surface 30 ofthe diffraction grating and the output slit 32 located in front of themirror and made similarly to the entry slit 25.

The electrodynamic driver 22 of the positional type has the motor 33(FIG. 12) comprising the stator (not shown in the FIG. 12) and the rotor34 fixed on the shaft 35 of the motor 33. Besides the driver 22 has thetransducer 36 of a turn angle of the rotor 34 located on one axis withthe motor 33 and representing an inductive differential converter of aturn angle of the rotor 34 into an electrical signal. The transducer 36has the modulator 37 fixed on the shaft 35 of the motor 33 with theeccentricity “e” concerning the rotation axis 38 of the rotor 34.

The rotor 34 of the motor 33 is designed as a ring consisting of a setof permanent magnets 39 and intermediate legs 40. Windings of the motor33 are formed by several coils 41 with the wire 42 located motionlesslyon the stator along the ring of the rotor 34. The ring of the rotor 34is rigidly connected to the shaft 35 of the motor 33 locatedperpendicularly to the plane of FIG. 12.

The turn angle transducer 36 contains two coils 43 placed on legs 44with pole tips 45 located diametrically opposite concerning themodulator 37, in addition the geometrical centre A of the modulator 37is displaced relatively to the common axis of rotation of the motor 33and the turn angle transducer 36 by the eccentricity “e”. The specifiedeccentricity “e” allows to realize the amplitude dependence of theoutput signal of the turn angle transducer 36 upon the turn angle of therotor 34 of the motor 33.

One optical element 26 or 27 or 28 of the monochromator 23 should befixed on the shaft 35 of the motor 33 of the electrodynamic driver 22with a possibility to turn around its own axis lying on the workingsurface of this element.

In FIG. 11 a mode is presented when the dispersive element 28, namely,the plane diffraction grating, is fixed on the shaft 35 of the motor 33.In addition any other element destined for similar purposes, forexample, the optical prism can be used as the dispersive element 28. Bythe dotted line in FIG. 11 the version is shown when the focusing mirror27 is fixed on the shaft 35 of the motor 33, thereat the dispersiveelement 28 and the second optical elements 26, namely, the collimatingmirror, should be fixed motionlessly.

The designed mode of the simple monochromator is possible when themonochromator contains fixed on the general basis the entry and outputslits, the collimating mirror and the dispersive element having aconcave working surface. The concave surface of the mirror is orientedin the direction of the entry slit and the working surface of thedispersive element which in this case can represent a concavediffraction grating, its concave surface is oriented simulteneously inthe direction of the output slit made similarly to the entry slit.

In the case when the light source 1 of light radiation forms a gatheringlight beam, it is possible even more simple mode of the simplemonochromator design when the monochromator contains fixed on thegeneral basis the entry and output slits and the dispersive element thatrepresents a concave diffraction grating, and its concave workingsurface is oriented simulteneously in the direction of the entry andoutput slits.

In other design mode of the present invention the selector 46 (FIG. 13)comprises a great number of optical elements 47 being a set ofnarrow-band interference filters 47, each of them has a passband in therange of the certain wavelength. The filters 47 are fixed on the shaft35 of the electrodynamic driver 22 by means of the cassette 48 with apossibility of their serial introduction into the light flow having thedirection shown in FIG. 13 by the arrow B. The quantity of filters 47depends on the amount of varieties of a biologically active substancethat are required to be determined by means of the offered device.

The device works as follows.

According to the method described above, a set of control samples withdifferent fixed concentrations of the certain substance to be analysedis prepared, and the measurement of signals of the circular dichroism iscarried out consistently at two wavelengths for each sample, onemeasurement is done at the wavelength 270 nm, and another one at thewavelength that is characteristic for the present analyzed substance.Using the received meanings, the signals of circular dichroism forcharacteristic wavelengths are normalized on the signals correspondingto the wavelength 270 nm, for every pair of meanings. The calculatednormalized values and the appropriate concentrations of the analyzedsubstance are written into a memory of the computer, and the diagram isbuilt of the dependence of the normalized signal upon the concentrationof the BAS, i.e. the calibration curve, that is marked in the computer.

Further, according to the method described above, the analyzed sample isprepared containing a mixture of the prepared before analyzed liquidcontaining the biologically active substance and of the lyotropicliquid-crystalline DNA dispersion formed in a polymer that is neutral inrelation to the DNA. After this procedure the sample is put into thecell 5 of the offered device, and the device is switched on.

The source 1 of the light radiation radiates a broad-band light flowthat enters the entry slit 25 of the selectors 2 of wavelengths, and thenarrow-band light flow with one known wavelength is radiated from theselector 2 through its output slit 32. This light flow passes throughthe polarizer 3, becomes linearly polarized with the certain directionof the polarization vector, gets then into the optical entry of themodulator 4 of polarization, passing through which it becomescircular-polarized with a periodically varied direction of rotation ofthe polarization vector rotating in the plane that is perpendicular tothe optical axis of the device. Then, passing the cell 5 with theanalyzed sample possessing the abnormal optical activity or, in otherwords, the circular dichroism, the light flow becomes modulated on itsintensity. Under action of light the electrical signal appears on theoutputs 7, 8 of the photodetector 6, and on the output 8 the variableproduct is registered that is proportional to ΔA (the value of thesignal caused by the abnormal optical activity), and on the output 7 thepermanent product is registered that is proportional to A (the value ofthe signal describing absorption of the biologically active substance ofthe sample), thereat the frequency of the variable product is equal tothe modulation frequency of light polarization. In the present devicethe permanent product is maintained at the fixed level by regulation ofthe power supply voltage of the photodetector 6, for that the signal ofthe permanent product from the output 7 of the photodetector 6 is put onthe entry of control of its power supply 9, i.e. a mode of stabilizationof the permanent product is realized by means of an inverse feedback onthe permanent product with simultaneous measurement of the variableproduct, that is equivalent to measurement of their ratio, and so, tomeasurement of the signal of circular dichroism of the sample to beanalysed. From the output 8 of the photodetector 6 the signal comes inthe first entry 10 of the synchronous amplifier 11, to the second entry21 of the photodetector 6 the reference signal is put at the frequencyof modulation of polarization. In the synchronous amplifier 11 thesignal is amplified, transformed into the direct current and directed tothe processing unit 14 for processing, where it is transformed into thedigital form, processed, compared with the calibration curve and put outas the meaning of the concentration in the sample of the biologicallyactive substance being under test. The control module 17 realizesnecessary interaction of all units of the device, realizes the requiredalgorithm of processing, produces the voltage with a modulationfrequency for work of the modulator 4 of polarization, forms thereference signal for functioning of the synchronous amplifier 11. Theway of processing the received signal in the digital form depends on thetype of the processing unit used for this purpose.

The electrodynamic driver 22 of the positional type of the selector 2works as follows.

The transducer 36 of a turn angle of the rotor 34 produces an electricalsignal, the amplitude of this signal depends on the turn angle of theshaft 35 of the motor 33 due to the presence of eccentricity “e” in aposition of the modulator 37 of the transducer 36 concerning the axis 38of rotation of the motor 33. At turn of the shaft 35 of the motor 33, abore between the pole tips 45 and the modulator 37 varies and, hence, anamplitude of the signal of the transducer 36 of a turn angle of therotor 34 varies as well. This signal enters the control module 17, whereits meaning is compared with the meaning that corresponds to the certainwavelength of the selector 2, and their difference is determined.Depending on this difference the control module 17 produces the controlsignal directed to the coils 41 of the stator of the motor 33. Themagnetic field caused by a current of the control signal flowing in thecoils 41 of the stator of the motor 33 interacts with the magnetic fieldof the permanent magnets 39 of the rotor 34 of the motor 33 in such amanner that the rotor 34, and so, the shaft 35 of the motor 33 turns onthe angle corresponding to the required wavelength of the selector 2, independence on the amplitude of the control signal.

In the case of usage of the monochromator 23 as the selector 2 theprinciple of its action does not differ from the principle of action ofwell known diffraction monochromators designed on the Czerny-Turnerscheme [M. Czerny, A. F. Turner and M. V. R. K. Murty. Principles ofMonochromators, Spectrometers and Spectrographs. Optical Engineering,vol. 13, N1, 1974, pp.23-38].

The difference is that the electrodynamic driver 22 of the positionaltype controlled by the signal coming from the control module 17 of thedevice is used as an arrangement for turn of one of optical elements 26,27, 28 of the monochromator 23 for wavelength tuning.

In one mode of the monochromator design described above and shown inFIG. 11, the light from the light radiation source 1 through the slit 25comes to the collimating mirror 26 that directs a parallel light beam tothe diffraction grating 28 which forms a light flow of the requiredwavelength. It after reflection from the concave surface of the focusingmirror 27 is focused on the output slit 32 and comes through it to thepolarizer 3. In another mode of the monochromator design describedabove, the light from the light radiation source 1 through the entryslit comes to the collimating mirror that directs a parallel light beamto the concave working surface of the diffraction grating. The lightflow of of the required wavelength is focused on the output slit andcomes through it to the polarizer 3.

In one more mode of the monochromator design, the light from the lightradiation source 1 through the entry slit comes to the concave surfaceof the diffraction grating. The light flow of the required wavelength isfocused on the output slit and comes through it to the polarizer 3.

In the case of usage of a set of the interference filters 47 as theselector 2 the device works as follows. The light flow from the lightradiation source 1 passes through one of the mentioned above filters 47located on a way of the light flow at the moment, and it is transformedon the output into the light flow with the required wavelength definedwith the passband of the present filter 47. At necessary changes of thewavelength, the control signal from the control module 17 enters themotor 33 of the electrodynamic driver 22 of the positional type causinga turn of the shaft 35 of the motor 33 on other angle, so another filter47 made for another wavelength is entered to the light flow, and theoutput light flow gets the wavelength defined by the passband of thisfilter 47.

Below examples of realization of the offered method with using theoffered device are shown.

EXAMPLE 6 Analytical Possibilities of the Device

To check the correctness of the CD measurements and the reception of theauthentic information about the optical properties of the analyzedliquids with the help of the offered device its calibration has beencarried out with the use of the aqueous solution of n-propylammoniumsalt of d-10-camphorsulphonic acid and the aqueous-salt solution of thelinear B-form double-stranded DNA.

6A. Calibration of the device with the use of the aqueous solution ofn-propylammonium salt of d-10-camphorsulphonic acid and the aqueous-saltsolution of the linear B-form double-stranded DNA.

6A.1. The aqueous solution of n-propylammonium salt ofd-10-camphorsulphonic acid (C₁₀H₁₆O₄S) is usually applied forcalibration of standard commercial dichrographs and spectropolarimeters.The CD spectrum of the aqueous solution of this acid (at certainconcentration and temperature) is characterized by the presence of thepositive band located in the UV-region of the spetrum (230-320 nm). Theshape of this band, the exact position of its maximum, the amplitude ofthis band are in details described [Gillon M. F., Williams R. E. (1975).Can. J. Chem., 53, pp.2351-2353].

For calibration of the device, the aqueous solution of C₁₀H₁₆O₄S withconcentration 0.15 mg/ml was used. For this purpose 2 ml of aqueoussolution of the specified concentration were placed into the rectangularoptical quartz cell of the device (optical path length=1 cm), and its CDspectrum in the region of wavelengths 250-350 nm was registered by thedevice.

The CD spectrum of the solution, observable on the screen of the monitorof the computer connected with the device, is shown in FIG. 14.

In the CD spectrum of the solution in the region of wavelengths 250-320nm the positive band is present, its amplitude and the maximum position(λ=290 nm) completely coincide with the literature data (see above).

6A.2. The well known in the literature conservative CD spectrum of thelinear B-form double-stranded DNA is characterized by the presence oftwo bands of different signs with the approximately equal amplitudes.The positive band has a maximum at λ˜278 nm, and the negative band—atλ˜247 nm. One more “control” point in the CD spectrum of the linearB-form double-stranded DNA is the wavelength, at which molecular opticalactivity of the DNA aqueous-salt solution, changing its sign, becomes“zero”. According to the literature data [Results of science andengineering (1975), vol. 1, ed. by Volkenstein M. V., VINITI, Moscow,p.115] this optical effect is observed at λ˜258 nm.

For calibration of the device, the aqueous-salt solution of the linearB-form double-stranded DNA (C_(DNA)˜5 μg/ml; 0.3 M NaCl+10⁻² M phosphatebuffer) prepared according to items 1.1, 1.2 and 1.4 was used.

2 ml of the aqueous-salt DNA solution was placed into the rectangularoptical quartz cell of the device (optical length 1 cm), and its CDspectrum in the region of wavelengths 240-310 nm was registered by meansof the device.

In FIG. 15 the CD spectrum of the solution, observable on the screen ofthe monitor of the computer connected with the portable device, ispresented.

Registered with the help of the device the CD spectrum (FIG. 15)completely repeats the above described peculiarities of the conservativeCD spectrum of the linear B-form double-stranded DNA.

Thus, the results received in item 6A testify reliability ofregistration of the optical properties of the analyzed solutions bymeans of the offered device.

6B. Determination of optical properties of the liquid-crystalline DNAdispersion and the liquid-crystalline dispersions of (DNA-MX) complexesby the offered device.

6B.1. 2 ml of the liquid-cryslalline DNA dispersion (C_(DNA)˜5 μg/ml;C_(PEG)=170 mg/ml; 0.3 M NaCl+10⁻² M phosphate buffer) preparedaccording to the example 1 are placed into the rectangular quartz cellof the device (optical length 1 cm), and its CD spectrum in the regionof wavelengths 240-310 nm is registered by means of the device.

In FIG. 16 the observable CD spectrum of the liquid-crystalline DNAdispersion is shown. This spectrum is characterized by the presence ofthe intensive negative band, its shape, sign, amplitude and maximumposition (λ˜270 nm) completely correspond to the data presented in FIG.2 (example 1) received by means of the standard laboratory “Jobin-Yvon”dichrograph.

6B.2. 4 μl of the solution prepared on item 2.1 are added to 2 ml of theliquid-crystalline DNA dispersion (item 6B.1) contained in the opticalcell of the device; the mixture obtained in the cell is stirred during30 s, and its CD spectrum in the region of wavelengths 580-720 nm isregistered by the offered device.

In FIG. 17 the CD spectrum of such the liquid-crystalline dispersiontreated with MX in the region of wavelengths from 580 up to 720 nm isshown. The addition of MX is accompanied by the appearance in the CDspectrum of the intensive negative band in the region of MX absorption,that is characterized by the presence of two maxima at λ˜680 nm andλ˜620 nm, that corresponds to the literature data. The appearance ofthis band testifies the formation of the liquid-crystalline dispersionfrom the (DNA-MX) complex (see p.18).

Thus, the results obtained convincingly testify that the device can beused for determination of MX in analyzed samples.

6C. Checking analytical possibilities of the device on theliquid-crystalline dispersions of the (DNA-MX) complexes.

6C.1. 2 ml of the liquid-crystalline DNA dispersion (C_(DNA)˜5 μg/ml;C_(PEG)=170 mg/ml; 0.3 M NaCl+10⁻² M phosphate buffer) are placed intothe rectangular quartz cell of the device (optical length 1 cm), and itsabnormal optical activity at λ=270 nm is registered by the device.

6C.2. 1 μl-portions of the aqueous-salt MX solution (C_(MX)=0.2 mg/ml(3.868×10⁻⁴ M); 0.3 M NaCl+10⁻² M phosphate buffer), altogether 4portions (the total volume of the added solution is 4 μl) are added to 2ml of the liquid-crystalline DNA dispersion (item 6C.1) contained in theoptical cell. After each added portion of MX the mixture obtained in theoptical cell is stirred (30 s), and its abnormal optical properties atλ=270 nm are registered by the device.

6C.3. The data received in the measurements at λ=270 nm (item 6C.2) arecompared with the results of the measurement carried out in item 6C.1.If the results of these measurements differ not more than 5%, one canbegin to registrate the optical signal at λ=680 nm generated by theliquid-crystalline DNA dispersion as a result of a complex formationwith MX molecules added (item 6C.2) to the solution.

6C.4. In complete agreement with items 6C.1-6C.3 one more titration ofthe liquid-crystalline DNA dispersion (item 6C.1) is produced with theaqueous-salt MX solution with smaller MX concentration (C_(MX)=0.02mg/ml (3.868×10⁻⁵ M; 0.3 M NaCl+10⁻² M phosphate buffer).

The results obtained during checking analytical possibilities of thedevice with the use of the liquid-crystalline dispersions of (DNA-MX)complexes have shown (FIG. 18) that, as well as in the case of the dataobtained with help of “Jobin-Yvon” dichrograph (FIG. 5, example 2), thedirectly proportional dependence is observed between the value of theoptical signal generated by the liquid-crystalline DNA dispersion atλ=680 nm at the formation of the (DNA-MX) complex and the Ct MX value.This dependence shows that due to the device one can determine thepresence and the concentration of MX in analyzed samples.

The check has shown that a large advantage of the device is that itallows quickly and precisely to determine MX in a wide range ofconcentrations including concentrations from 5×10⁻⁷ down to 5×10⁻⁸ M,i.e. one order of magnitude lower than limit concentrations detected bythe known devices of “Jobin-Yvon” and “Jasco” firms.

Thus, the offered method and the offered device allow quickly, preciselyand with a high sensitivity to determine the presence and theconcentrations of BAS (MX) in blood of patients, whose therapy isconnected with application of antitumor compounds.

The offered method excludes the use of complex and expensive equipmentand the presence of a highly skilled personnel as well, and can be usedfor determination of other biologically active and pharmacologicalcompounds forming intercalation complexes with DNA bases pairs.

INDUSTRIAL APPLICABILITY

The offered invention can be used in medical and clinical biochemistry,and also for molecular pharmacology at research of pharmaco-kinetics ofbiologically active substances, for pharmaceutical industry and ecology.The most effective its use is in clinical biochemistry.

What is claimed is:
 1. The method for determination in an analyzedliquid of a biologically active substance interacting with a lyotropiccholesteric liquid-crystalline DNA dispersion formed in a polymerneutral in respect to DNA, distinguished by: that the lyotropiccholesteric liquid-crystalline dispersion is formed in an aqueous-saltsolution of the specified polymer of the linear double-stranded DNAmolecules of a low molecular mass immediately before mixing with theanalyzed liquid containing the determined biologically active substance,thereat the analysed liquid is previously mixed with the specifiedpolymer under conditions, when the optical properties of the lyotropicliquid-crystalline DNA dispersion are not broken, then through theanalysed sample, obtained as a result of mixing of the prepared analyzedliquid with the specified liquid-crystalline DNA dispersion, a flow ofcircular-polarized light is passed, and the optical signal is registeredgenerated by the liquid-crystalline dispersion at two wavelengths, oneof them is in the region of the DNA absorption, and another one is inthe region of absorption of the determined biologically activesubstance, then the ratio is calculated between these signals at thespecified wavelengths, and the concentration of the biologically activesubstance is determined on this ratio using the calibration curve. 2.The method on item 1, distinguished by that polyethyleneglycol is usedas the neutral polymer.
 3. The method on item 1, distinguished by that abiological liquid is used as the analyzed liquid.
 4. The method on item3, distinguished by that blood is used as the biological liquid.
 5. Themethod on item 3, distinguished by that plasma of blood is used as thebiological liquid.
 6. The method on item 1, distinguished by that thebiologically active substance represents an antitumor substance of theanthraquinone group.
 7. The method on item 6, distinguished by that theantitumor substance of the anthraquinone group represents amithoxantrone.
 8. The method on item 3, distinguished by that atdetermination of the biologically active substance in the biologicalliquid with heterogeneous in it distribution of the biologically activesubstance, its concentration obtained due to the calibration curve iscorrected in view of factor of its distribution in the biologicalliquid.
 9. The device for determination of a biologically activesubstance in an analyzed liquid, comprising installed consistently:Source (1) of light radiation; Selector (2) having at least one opticalelement (26, 27, 28, 47) and forming a light flow of a certainwavelength; Polarizer (3) forming a linearly polarized light flow of thespecified light flow; Modulator (4) of polarization transforming thelinearly polarized light flow into a circular-polarized light flow witha periodically varied direction of rotation of its polarization vector;Cell (5) for an analysed sample; Photodetector (6) transforming theoptical signal generated by components of the sample to be analyzed intothe proportional electrical signal; Synchronous amplifier (11)increasing the pointed electrical signal; Processing unit (14) forprocessing the received electrical signal and for calculation ofconcentration of the biologically active substance; Control module (17),distinguished by that the selector (2) contains an electrodynamic driver(22) of a positional type, designed with a possibility of setting atleast two wavelengths, at which the optical signal generated by thecomponents of the sample under analysis is registered, and at least oneoptical element (26, 27, 28, 47) fixed on a motor (33) shaft (35) of thespecified driver (22).
 10. The device on item 9, distinguished by thatthe selector (2) represents a simple monochromator (23) containing atleast two optical elements (26, 28), one of them represents a dispersiveelement (28), and one of these optical elements (26 or 28) is fixed onthe motor (33) shaft (35) of the specified driver (22) with thepossibility to turn around its own axis.
 11. The device on item 10,distinguished by that the dispersive element (28) represents adiffraction grating.
 12. The device on item 11, distinguished by thatthe diffraction grating (28) is designed concave.
 13. The device on item9, distinguished by that the selector (2) represents a simplemonochromator (23) containing one optical element (28) fixed on themotor (33) shaft (35) of the specified driver (22) with the possibilityto turn around its own axis and representing the dispersive element (28)designed as the concave diffraction grating.
 14. The device on item 9,distinguished by that the selector (2) contains a grate number ofoptical elements (47) being a set of narrow-band interference filters(47) fixed on the motor (33) shaft (35) of the specified driver (22)with a possibility of their alternate introduction into the light flow,in addition each of these filters (47) has a transparency band in theregion of the certain wavelength chosen for the concrete biologicallyactive substance.
 15. The device on item 9, distinguished by that theelectrodynamic driver (22) contains the transducer (36) of a turn angleof its motor (33) rotor (34), representing an inductive differentialconverter of a turn angle of the rotor (34) into an appropriateelectrical signal, and containing a modulator (37) designed as a ringfixed on the motor (33) shaft (35) with an eccentricity (“e”) concerningits rotation axis (38).